Capillary electrophoresis (CE) has proven to be a powerful technique for analysis of complex mixtures of biological molecules, offering advantages not available with conventional separation methods, such as HPLC or column chromatography. One advantage of CE is the speed of the analysis. Frequently, a sample can be analyzed by CE in a span of time on the order of minutes, whereas conventional techniques often require several hours or more. The low volume of sample required is another advantage of this technique. Samples of only a few nanoliters are easily analyzed by CE; other separation techniques usually require samples of more than one .mu.l. The low CE sample size requirement allows the remainder of the sample volume to be used for other manipulations or further analyses. An additional advantage is the possibility for high resolution of the sample into its component parts with component molecules separated by CE eluting in very narrow bands. This capability allows the user to analyze a sample of very similar molecules, e.g., DNA fragments with a single base change or proteins with a single amino acid substitution.
Although the advantages of capillary electrophoresis allow an investigator to process a very small sample, current methods of collecting or removing the separated components are limited by the small volumes (approximately a few nL) of fractionated materials that elute from the capillary tube and the imprecise way the samples are usually collected. Available sample collection devices for use in CE generally use electroelution (including the movement of ions with or without electroosmotic flow) or pressurized flow.
Electroelution methods of sample collection employ standard capillary electrophoretic equipment. The user must know several variables of the system including the velocity of migration of a selected sample, and the distance between the on column detection point and the end of the capillary. After detection of a zone of interest, the time required for the zone to traverse the distance between the detection point and the capillary exit is calculated. In one general system, the electric current is then turned off, and the CE capillary is removed from the CE apparatus and placed into a collection vial containing a collection buffer and a platinum electrode. To collect the zone, current is applied for a predetermined time so that the zone migrates from the capillary into the collection vial. In some cases, pressure can be applied to remove the sample from the capillary. After collection of the zone, the capillary is moved back to the electrode reservoir and the analysis can continue. Alternatively, a collection capillary containing a collection buffer and electrode can be positioned adjacent to the exit end of the capillary while the electric current is off. The current is then resumed for collection of the zone of interest.
Electroosmotic elution can be used in cases when electroosmotic flow is present in the electrophoretic system. The end of the capillary used in this approach has a microscopic fracture located after the detection point. The microscopic fracture is immersed in the electrode buffer reservoir to provide the electric contact for completion of the circuit. As electroosmotic flow transports the liquid inside the CE capillary, the separated zones move past the fracture and most of the material can be collected in appropriate vials once the time of their appearance at the capillary exit is calculated.
Capturing segregated samples on membranes represents another approach for collecting samples from a capillary electrophoretic separation. In this approach, the exit end of the capillary is in contact with a wetted surface of a moving membrane (e.g., cellulose acetate, polyvinylidene difluoride (PVDF), or nitrocellulose) which is connected to the electrode. As the separated zones elute from the capillary, they are deposited on the surface of the membrane. The collected fractions are not obtained in a solution form, and their location on the support can be difficult without further sample treatment (blotting, staining).
It is also known to combine CE directly with other detection systems, e.g., mass spectroscopy. In one such system, the sample components emerging from the outlet end of the capillary enter an electrospray ionization (ESI) interface. Sample ions in the gas phase are then transferred to a mass spectrometer for analysis. To increase resident dwell time in the mass spectrometer, step changes can be introduced in the electric field strength of the CE system. For example, prior to elution of the first sample component from the capillary under constant field strength conditions, the electrophoretic voltage can be decreased. This reduction of migration velocity of the emerging components permits more efficient ionization at the ESI interface and an increased number of mass spectroscopy scans can be recorded without a significant loss in ion intensity.
Additional methods of processing the separated components of a sample following CE would be desirable to take full advantage of the high resolution capability of this separation technique. The capillary electrophoretic system and method of the invention provide for an improved interface between a separation capillary and subsequent systems of sample collection or analysis.